Embodiments of the present invention relate to confocal microscopy with fluorescence to support surgical procedures, such as incremental surgery to remove tumors that are not naturally visible to a surgeon. In some embodiments, multimodal confocal microscopy is used to render images that emulate frozen histology sections.
Several surgical procedures to remove tumors involve cell morphology that is difficult to discern with the unaided eye. In such circumstances, successive incremental excisions are performed, and each sample of excised tissue is subjected to histological examination to determine the remaining extent of the tumor. The position and extent of the tumor evident in the histology sections is used to guide the next excision. However, the procedure for preparing the histology sections is tedious and time consuming, often exceeding 30 minutes per section. Thus, surgical procedures that rely on such histology sections can extend to two hours and more, increasing the exposure of patient to infection and undesirable consequences of surgery, and limiting the availability of surgeons for other procedures.
For example, Mohs surgery for the removal of basal cell carcinoma (BCC) in skin often requires several excisions. After each excision, a frozen histology section is prepared while the surgeon waits. The location and extent of the tumor evident in the frozen histology section guides the next excision. The preparation of the frozen histology section, including staining to enhance visibility of certain structures, is tedious and takes 24 to 40 minutes. Thus the surgery usually lasts several hours.
Other surgical procedures that involve viewing histology of incremental excisions include the removal of oral mucosal lesions, thyroid nodules, parathyroid glands and bone, and include needle core biopsies and lumpectomies of the breast, and inter-operative biopsies of liver and bladder, among others.
Confocal microscopy is capable of directly observing, in real time, very small structures in a very small field of view and can directly observe tumors in excised tissue. A confocal microscope achieves very high resolution by using the same objective lens to focus both a parallel beam of incident light and the resulting emitted light at the same small spot on or near the surface of target tissue. For example, a typical confocal microscope using an objective lens that magnifies an object in the focal plane 30 times (30×) resolves spots that are about half of a micron (μm, 1 μm=10−6 meters) across, sufficient to resolve morphology of a nucleus of a cell, which is about 10 microns. A field of view is obtained by scanning the confocal spot across the tissue by changing the angle of the incident beam of light. In an example confocal microscope, the field of view is about 400 μm, and includes about 1024 rows and 1,024 columns of pixels, for an individual image of about one million pixels.
This confocal microscope field of view is small compared to the field of view of histology sections. A histology section often includes a field of view that is millimeters to tens of millimeters (mm, 1 mm=10−3 meters) across, about forty times larger than the confocal microscope field of view. For example, in Mohs surgery, a 2× magnification is used in a standard light microscope to view a 12 mm by 12 mm (12×12 mm) portion of the frozen histology section.